Acta Agrestia Sinica ›› 2014, Vol. 22 ›› Issue (4): 911-914,918.DOI: 10.11733/j.issn.1007-0435.2014.04.037

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Optimizing ISSR-PCR System of Carex atrofusca Using Orthogonal Design

HU Yan-ping1, WANG Li1, BAO Rui1, SHI Lin1,3, XU Rong-hua2, LI Yi1   

  1. 1. Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining, Qinghai Province 810008, China;
    2. Haixi Science and Technology Bureau, Delingha, Qinghai Province 817000, China;
    3. University of Chinese Academy of Sciences, Beijing 100049, China
  • Received:2013-11-13 Revised:2014-02-17 Online:2014-08-15 Published:2014-08-04

正交设计优化黑褐苔草ISSR-PCR反应体系

胡延萍1, 王莉1, 包蕊1, 石琳1,3, 旭荣花2, 李毅1   

  1. 1. 中国科学院西北高原生物研究所, 青海 西宁 810008;
    2. 海西州科学技术局, 青海 德令哈 817000;
    3. 中国科学院大学, 北京 100049
  • 通讯作者: 李毅
  • 作者简介:胡延萍(1981-),女,山东菏泽人,博士,副研究员,研究方向为植物生物技术,E-mail:yphu@nwipb.cas.cn
  • 基金资助:

    国家科技支撑计划(2012BAC08B04);国家自然科学基金(31200245);2011年中国科学院“西部之光”人才培养计划“西部博士资助”项目资助

Abstract:

An orthogonal design was used to optimize the ISSR-PCR system of Carex atrofusca at four levels of five factors (Mg2+, Taq DNA polymerase, dNTP, primer and DNA template). Then, based on the optimal ISSR-PCR system, the annealing temperature was selected. The optimal PCR (20 μL) mixture contained 1.80 mmol·L-1 Mg2+, 0.60 U Taq DNA polymerase, 0.125 mmol·L-1 dNTP, 0.3 μmol·L-1 primer and 50 ng template DNA. The suitable annealing temperature of primer UBC807 was 55℃. With the optimal system of ISSR-PCR, clear and steady bands were obtained in different individuals of C. atrofusca.

Key words: Carex atrofusca, ISSR-PCR, Reaction conditions, Orthogonal design

摘要:

采用正交设计对黑褐苔草(Carexa trofusca)ISSR-PCR反应的Mg2+Taq DNA聚合酶、dNTP、引物和DNA模板进行优化,以期建立最佳反应条件并筛选退火温度。结果表明:黑褐苔草20μLISSR-PCR最佳反应体系为:1.80μmol·L-1 Mg2+、0.60 U Taq DNA聚合酶、0.125μmol·L-1 dNTP、0.3μmol·L-1 Primer和50ngDNA模板;引物UBC807适宜退火温度为55℃。该体系能在不同个体中扩增出清晰的、稳定性好的条带。

关键词: 黑褐苔草, ISSR-PCR, 反应体系, 正交设计

CLC Number: